Abstract
Intrinsically disordered and phenylalanine-glycine rich nucleoporins (FG-Nups) form a selective permeability barrier inside the nuclear pore complex (NPC): Large molecules can only cross the central channel of the NPC when piggybacked by nuclear transport receptors (NTRs) that specifically interact with FG-Nups. These FG-Nups, however, display complex and non-random amino acid architecture and possess repeatedly occurring FG-motifs flanked by distinct amino acid stretches. How such heterogeneous sequence composition relates to function and how homotypic interactions between such disordered stretches, and transient heterotypic interactions with folded transport receptors could give rise to a transport mechanism is still unclear. We have now developed an integrated chemical biology-fluorescence approach to study the molecular plasticity of FG-Nups on the single-molecule level using multi-parameter fluorescence spectroscopy. Despite its heterogeneous primary sequence, the unstructured FG-domain of Nup153 displays a collapsed coil behavior across its entire amino acid sequence, due to favorable intrachain interactions. We show that those interactions at the dynamic disordered state can induce aggregation and lead to the formation of stable amyloid fibers that, at high protein concentrations, can further enlace to form macroscopic hydrogels with NPC like properties. Amyloid formation can also be inhibited by the presence of NTRs. Furthermore, we found that Nup153 retains its collapsed conformation even when involved in NTR•Nup complexes. Using fluorescence lifetime and polarized fluorescence fluctuation analysis with picosecond resolution, we were able to observe the formation of very flexible and dynamic complexes and detect novel binding modes underlying the nucleocytoplasmic transport process. Synergistic molecular dynamics simulations permit visualization of previously unknown steps and determinants during Nup•NTR interactions at atomic resolution. These results provide important insights on how nuclear transport can pursue specifically and very fast inside the nuclear pore complex.
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